Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis.

Regulation of apoptosis is crucial for tissue homeostasis under normal development and environmental stress. In Drosophila, cell death occurs in different developmental processes including embryogenesis. Here, we report that two members of the miR-2 seed family of microRNAs, miR-6 and miR-11, function together to limit the level of apoptosis during Drosophila embryonic development. Mutants lacking both miR-6 and miR-11 show embryonic lethality and defects in the central nervous system (CNS). We provide evidence that miR-6/11 functions through regulation of the proapoptotic genes, reaper (rpr), head involution defective (hid), grim and sickle (skl). Upregulation of these proapoptotic genes is responsible for the elevated apoptosis and the CNS defects in the mutants. These findings demonstrate that the activity of the proapoptotic genes is kept in check by miR-6/11 to ensure normal development.

AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin.

The AP-2 family of transcriptional regulator proteins has three members, alpha, beta and gamma. AP-2alpha and gamma are expressed in placenta and in the human trophoblast cell line JEG-3. AP-2 has been shown to regulate expression of the placental human chorionic gonado-tropin (hCG) alpha- and beta-subunit genes, however, previous work did not distinguish between the family members. Tryptic peptides of the AP-2 protein complexes purified from JEG-3 cells by oligo-affinity chromatography using the hCGalpha AP-2 site match the amino acid sequence of AP-2gamma. The fact that AP-2gamma is present at significant levels and binds the hCGalpha trophoblast-specific element suggests that AP-2gamma is at least part of the binding complex in vivo and plays a role in regulating hCG expression. We show that mutation of each of four AP-2 binding sites within the hCGbeta promoter decreases expression in transfection assays, demonstrating that all four sites are required for maximal expression in JEG-3 cells. Furthermore, we find differences in regulation of the family members: AP-2alpha mRNA levels increase in response to cAMP while AP-2gamma mRNA levels do not. The demonstrated importance of the AP-2 sites in controlling hCGalpha and beta expression and the likely involvement of more than one family member suggest that a balance in AP-2 proteins is involved in coordinate regulation of these genes. Moreover, many placenta-restricted genes are regulated by AP-2 proteins, thus members of this family may play an important overall role in placenta-specific expression.

Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells.

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. In the embryonic central nervous system of Drosophila melanogaster, secondary precursor cells-ganglion mother cells (GMCs)-divide and produce postmitotic neurons that take on different cell fates. In this study, we show that binary fate decision of two pairs of sibling neurons is accomplished through the interplay of Notch (N) signaling and the intrinsic fate determinant Numb. We show that GMCs have apical-basal polarity and Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. Our results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, our data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells.

Mutations in lottchen cause cell fate transformations in both neuroblast and glioblast lineages in the Drosophila embryonic central nervous system.

The Drosophila embryonic central nervous system (CNS) develops from a stereotyped pattern of neuronal progenitor cells called neuroblasts (NB). Each NB has a unique identity that is defined by the time and position of its formation and a characteristic combination of genes it expresses. Each NB generates a specific lineage of neurons and/or glia. Here we describe the genetic and phenotypic analysis of lottchen (ltt), a novel gene whose loss of function causes a change in the identity of at least one NB as well as cell fate transformations within the lateral glioblast lineage. In wildtype embryos the parental NB of the motoneuron RP2 is NB4-2. ltt embryos are distinguished by an additional RP2-like neuron which appears later in development. We show that the two RP2 neurons are derived from two distinct GMC4-2a-like cells that do not share the same parental NB, indicating that a second NB has acquired the potential to produce a GMC and a neuron which is normally restricted to the NB4-2 lineage. Moreover, the ltt mutations lead to a loss of correctly specified longitudinal glia; this coincides with severely defective longitudinal connectives. Therefore, lottchen plays a role in specifying the identity of both neuroblast and glioblast lineages in the Drosophila embryonic CNS. We discuss the possibility that ltt may act to differentiate NB identity along the medial lateral axis.

Evaluation of controlled-release mosquito repellent formulations.

Eight polymer and 9 microcapsule formulations of deet were tested on laboratory rabbits against Aedes aegypti and Anopheles albimanus. Several formulations were significantly more effective than simple (unformulated) deet at the same strength for periods up to 24 h. Best results were obtained with a polymer formulation containing a high molecular weight fatty acid and 3 microcapsule formulations containing lanolin, gum arabic, gelatin, tannic acid, stearic acid, polypropylene glycol, water, and a commercial lotion in the microcapsule and carrier fractions.

Evaluation of the laboratory rabbit model for screening topical mosquito repellents.

The laboratory rabbit was evaluated as a model for screening topical mosquito repellents, using data obtained in tests of deet (N,N-diethyl-3-methylbenzamide) against Aedes aegypti on humans and rabbits. Host-specific differences in the action of the test material were quantified by multiple regression analysis. The test material was less effective but more persistent in tests on rabbits, and responses of the mosquito test population were more variable.

Evaluation of the laboratory mouse model for screening topical mosquito repellents.

Eight commercial repellents were tested against Aedes aegypti 0 and 4 h after application in serial dilution to volunteers and laboratory mice. Results were analyzed by multiple regression of percentage of biting (probit scale) on dose (logarithmic scale) and time. Empirical correction terms for conversion of values obtained in tests on mice to values expected in tests on human volunteers were calculated from data obtained on 4 repellents and evaluated with data obtained on 4 others. Corrected values from tests on mice did not differ significantly from values obtained in tests on volunteers. Test materials used in the study were dimethyl phthalate, butopyronoxyl, butoxy polypropylene glycol, MGK Repellent 11, deet, ethyl hexanediol, Citronyl, and dibutyl phthalate.

Cushing syndrome in pregnancy.

The occurrence of pregnancy in the face of untreated Cushing syndrome is rare because of the high incidence of ovulatory disturbances experienced by patients with the disorder. A total of 58 patients with 65 pregnancies has been reported in the literature to date. Although pituitary-dependent adrenal hyperplasia is the most common etiology of Cushing syndrome in the general population, adrenal adenoma is more common in the pregnant population. Significant maternal morbidity is attributable to hypertension, congestive heart failure, and poor tissue healing. Prematurity and intrauterine growth retardation account for most of the perinatal morbidity; perinatal mortality is substantial. Treatment directed at relieving hypercortisolism has been instituted during pregnancy: Pituitary or adrenal surgery, chemotherapy, and pituitary irradiation have all been reported, with variable results. Information is lacking on any alteration of maternal morbidity after treatment. The impact of therapy on perinatal outcome appears limited to a reduction in the prematurity rate, but overall numbers are small and such a conclusion should be viewed with caution. No significant maternal or perinatal complications secondary to treatment itself were reported.

A case report of prostaglandin E2 termination of pregnancy complicated by Cushing's syndrome.

A case of Cushing's syndrome caused by an adrenal adenoma seen at 10 weeks' gestation is described. The pregnancy was terminated at 18 weeks' gestation, representing the first reported case of therapeutic termination of pregnancy in Cushing's syndrome with prostaglandin E2 vaginal suppositories.

Attraction of mosquitoes to diethyl methylbenzamide and ethyl hexanediol.

Studies by prior workers have shown that insect repellents can act as attractants when present as low concentrations, deposits or residues. In the present study deet and ethyl hexanediol were tested in 2-fold serial doses from 1.9 X 10(-9) to 1.6 X 10(-2) mg/cm2 on the forearms of volunteers against colonized Anopheles albimanus, Aedes aegypti and Ae. taeniorhynchus. Both compounds were significantly repellent at the high end of the dose range, as expected. Neither was significantly attractant to An. albimanus in low doses. However, deet was significantly attractant to Ae. aegypti in the dose range 7.6 X 10(-9) to 1.2 X 10(-4) mg/cm2 and to Ae. taeniorhynchus in the dose ranges 1.9 X 10(-9) to 3.1 X 10(-8) mg/cm2 and 2.0 X 10(-6) to 2.5 X 10(-4) mg/cm2. Ethyl hexanediol was significantly attractant to Ae. taeniorhynchus in the dose range 1.9 X 10(-9) to 6.2 X 10(-5) mg/cm2. Based on these results and prior work of V.G. Dethier and C.N.E. Ruscoe, a model sequence of the effects of chemicals on insects with increasing dose was developed. It was concluded that the labels of commercial repellents should be amended to include instructions to wash off or reapply the repellent when it is no longer effective.

Mathematical models of the effectiveness and persistence of mosquito repellents.

Two models of the effectiveness and persistence of mosquito repellents on the skin were developed from published functions and data. The probit plane model, Y = a + b1X1 + b2X2, relates the response (Y, in probits) of the mosquito test population to the log dose (X1) of repellent applied and the test period, or elapsed time from the time of application (X2). The exponential decay model, Z = X1 + (b2/b1)X2, estimates the repellent residue (log-1 Z) from X1 and X2. The models were validated with original data from tests of deet (N,N-diethyl-3-methylbenzamide) and ethyl hexanediol (2-ethyl-1,3-hexanediol) on the forearm against the yellow fever mosquito, Aedes aegypti. The probit plane model was evaluated as Y = 8.83 + 1.56 X1-0.69X2 for deet and Y = 8.67 + 1.68X1 - 0.92X2 for ethyl hexanediol when X1 is in log mg/cm2 and X2 is in hours. The exponential decay model was evaluated as Z = X1 - 0.45X2 for deet and Z = X1 - 0.55X2 for ethyl hexanediol. The decay constant (lambda) and half-life (t1/2) were estimated as 1.03 hr-1 and 0.67 hr for deet and 1.26 hr-1 and 0.55 hr for ethyl hexanediol from the slope parameter (b2/b1) of the decay model. Applicable correlation coefficients, standard errors and confidence limits are given. The introduction of these models of the pharmacodynamics of mosquito repellents is a step toward establishing a rational basis for the research, development, testing and evaluation of repellents and for their regulation by the government.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical mapping of human adenovirus type 7 DNA using a simple and sensitive method of terminal labeling.

The tyrosine-containing peptide covalently attached to each 5'-terminus of adenovirus type 7 (Ad 7; Greider) DNA was labeled with 125I. The 5'-labeled DNA was subjected to digestion with several restriction endonucleases and the size of the labeled terminal fragments was determined. Partial hydrolysis by these endonucleases generated a series of labeled fragments which were fused to the terminal fragments and could, therefore, be detected by autoradiography. From the sizes of the partial products the location of the cleavage sites of the enzymes on Ad7 DNA could be determined. The subgenomic DNA extracted from incomplete particles by protease treatment could also be labeled with 125I, since it was found to contain the tyrosine-containing peptide covalently attached to the preferentially packaged left end of the genome.